We have found that 17b-Estradiol Dehydrogenase (17-ED) and 20a-Hydroxysteriod Dehydrogrenase (20-HSD) activites co-purify from the cytosol of human term placenta. Further, placental fractionation studies localize both activities to the 105,000 x g cytosol. Finally, affinity alkylation studies using 16a-bromoacetoxyprogesterone suggest that both oxidoreductase activities exist at one active site on a single protein. Thus, we hypothesize that steroid 17b- and 20a-oxidoreduction are effected at a single catalytic (active) site on a single enzyme. Affinity alkylating analogs and enzyme-generated affinity alkylators ("suicide substrates") of the estrene (18 carbon) and pregnene (21 carbon) series, and affinity labeling nucleotide analogs will be used with homogeneous enzyme to explore: 1. Whether both enzyme activities are simultaneously and identically modified during experimental conditions calculated to inactivate and reactivate the enzyme(s). 2. Whether corresponding "affinity radiolabeling probes" map identical amino acids within the enzyme active center because both the estrogen and progestin analogs are indeed interacting at a common locus on one protein. These studies will allow affirmation or denial of the bifunctional enzyme activity thesis. The proposed studies will structurally characterize enzyme(s) which, by regulating steroid hormone levels within the fetal-placental-uterine unit, are believed to participate in the events which initiate labor. They will further elucidate structural components which permit binding and catalysis of substrates at an enzyme active center. If 17-ED and 20-HSD activities in human placental cytosol do indeed reside at a single site, this will provide the first well-documented evidence in man for bifunctional, stereospecific oxidoreduction of steriod hormones.